Polycistronic expression vector construction

ABSTRACT

A polycistronic construction of xenogeneic sequences expressible in eukaryotic cells is described. DHFR encoding sequences under control of the same promoter with another foreign gene can be amplified with methotrexate, and thus cause coamplification of the foreign gene.

BACKGROUND OF THE INVENTION

This invention relates to the application of recombinant DNA technologyto the production of polypeptide in vertebrate cell cultures. Morespecifically, this invention relates to utilizing the coding sequencefor a secondary control polypeptide as a tool in controlling productionof a foreign polypeptide by the vertebrate cell culture.

The general principle of utilizing a host cell for the production of aheterologous protein--i.e., a protein which is ordinarily not producedby this cell--is well known. However, the technical difficulties ofobtaining reasonable quantities of the heterologous protein by employingvertebrate host cells which are desirable by virtue of their propertieswith regard to handling the protein formed are many. There have been anumber of successful examples of incorporating genetic material codingfor heterologous proteins into bacteria and obtaining expressionthereof. For example, human interferon, desacetyl-thymosin alpha-1,somatostatin, and human growth hormone have been thus produced.Recently, it has been possible to utilize non-bacterial hosts such asyeast cells (see, e.g., co-pending application, U.S. Ser. No. 237,913,filed Feb. 25, 1981;) and vertebrate cell cultures (U.S. applicationSer. No. 298,235, filed Aug. 31, 1981) as hosts. The use of vertebratecell cultures as hosts in the production of mammalian proteins isadvantageous because such systems have additional capabilities formodification, glycosylation, addition of transport sequences, and othersubsequent treatment of the resulting peptide produced in the cell. Forexample, while bacteria may be successfully transfected and caused toexpress "alpha thymosin", the polypeptide produced lacks the N-acetylgroup of the "natural" alpha thymosin found in mammalian system.

In general, the genetic engineering techniques designed to enable hostcells to produce heterologous proteins include preparation of an"expression vector" which is a DNA sequence containing,

(1) a "promoter", i.e., a sequence of nucleotides controlling andpermitting the expression of a coding sequence;

(2) a sequence providing mRNA with a ribosome binding site;

(3) a "coding region", i.e., a sequence of nucleotides which codes forthe desired polypeptide; and

(4) a "termination sequence" which permits transcription to beterminated when the entire code for the desired protein has been read;and

(5) if the vector is not directly inserted into the genome, a "replicon"or origin of replication which permits the entire vector to bereproduced once it is within the cell.

In the construction of vectors in the present invention, the samepromoter controls two coding sequences, one for a desired protein, andthe other for a secondary protein. Transcription termination is alsoshared by these sequences. However, the proteins are produced indiscrete form because they are separated by a stop and starttranslational signal.

Ordinarily, the genetic expression vectors are in the form of plasmids,which are extrachromosomal loops of double stranded DNA. These are foundin natural form in bacteria, often in multiple copies per cell. However,artificial plasmids can also be constructed, (and these, of course, arethe most useful), by splicing together the four essential elementsoutlined above in proper sequence using appropriate "restrictionenzymes". Restriction enzymes are nucleases whose catalytic activity islimited to lysing at a particular base sequence, each base sequencebeing characteristic for a particular restriction enzyme. By artfulconstruction of the terminal ends of the elements outlined above (orfractions thereof) restriction enzymes may be found to splice theseelements together to form a finished genetic expression vector.

It then remains to induce the hose cell to incorporate the vector(transfection), and to grow the host cells in such a way as to effectthe synthesis of the polypeptide desired as a concomitant of normalgrowth.

Two typical problems are associated with the above-outlined procedure.First, it is desirable to have in the vector, in addition to the fouressential elements outlined above, a marker which will permit astraightforward selection for those cells which have, in fact, acceptedthe genetic expression vector. In using bacterial cells as hosts,frequently used markers are resistance to an antibiotic such astetracycline or ampicillin. Only those cells which are drug resistantwill grow in cultures containing the antibiotic. Therefore, if the cellculture which has been sought to be transfected is grown on a mediumcontaining the antibiotic, only the cells actually transfected willappear as colonies. As the frequency of transformation is quite low(approximately 1 cell in 10⁶ being transfected under ideal conditions)this is almost an essential prerequisite as a practical matter.

For vertebrate cells as hosts, the transformation rate achieved is moreefficient (about 1 cell in 10³). However, facile selection remainsimportant in obtaining the desired transfected cells. Selection isrendered important, also, because the rate of cell division is aboutfifty times lower than in bacterial cells--i.e., although E. coli divideonce in about every 20-30 minutes, human tissue culture cells divideonly once in every 12 to 24 hours.

The present invention, in one aspect, addresses the problem of selectingfor vertebrate cells which have taken up the genetic expression vectorfor the desired protein by utilizing expression of the coding sequencefor a secondary protein, such, for example, as an essential enzyme inwhich the host cell is deficient. For example, dihydrofolate reductase(DHFR) may be used as a marker using host cells deficient in DHFR.

A second problem attendant on production of polypeptides in a foreignhost is recovery of satisfactory quantities of protein. It would bedesirable to have some mechanism to regulate, and preferably enhance,the production of the desired heterologous polypeptide. In a secondaspect of the invention, a secondary coding sequence which can beaffected by externally controlled parameters is utilized to allowcontrol of expression by control of these parameters. Furthermore,provision of both sequences on a polycistron in itself permits selectionof transformants with high expression levels of the primary sequence.

It has been shown that DHFR coding sequences can be introduced into,expressed in, and amplified in mammalian cells. Genomic DNA frommethotrexate resistant Chinese Hamster Ovary (CHO) cells has beenintroduced into mouse cells and results in transformants which are alsoresistant to methotrexate (1). The mechanism by which methotrexate (MTX)resistance in mouse cells is developed appears to be threefold: throughgene amplification of the DHFR coding sequence (2, 3, 4); throughdecrease in uptake of MTX (5, 6) and through reduction in affinity ofthe DHFR produced for MTX (7).

It appears that amplification of the DHFR gene through MTX exposure canresult in a concommitant amplification of a cotransfected gene sequence.It has also been shown that mouse fibroblasts, transfected with both aplasmid containing hepatitis B DNA sequences, and genomic DNA from ahamster cell line containing a mutant gene for MTX-resistant DHFR,secrete increased amounts of hepatitis B surface antigen (HBsAg) intothe medium when MTX is employed to stimulate DHFR sequence amplification(8). Further, mRNA coding for the E. coli protein XGPRT is amplified inthe presense of MTX in CHO cells co-transfected with the DHFR and XGPRTgene sequences under control by independent promoters (9). Finally,increased expression of a sequence endogenous to the promoter in aDHFR/SV40 plasmid combination in the presence of MTX has beendemonstrated (10).

SUMMARY OF THE INVENTION

The present invention is based on the discovery that, in vertebrate cellhosts, where the genetic expression vector for a desired polypeptidecontains a secondary genetic coding sequence under the control of thesame promoter, this secondary sequence provides for a convenientscreening marker, both for transformants in general, and fortransformants showing high expression levels for the primary sequence,as well as serving as a control device whereby the expression of adesired polypeptide can be regulated, most frequently enhanced.

This is particularly significant as the two proteins, according to themethod of this invention, are produced separately in mature form. Whileboth DNA coding sequences are controlled by the same transcriptionalpromoter, so that a fused message (mRNA) is formed, they are separatedby a translational stop signal for the first and start signal for thesecond, so that two independent proteins result.

As a vertebrate host cell culture system is often advantageous becauseit is capable of glycosylation, phosphorylation, and lipid associationappropriate to animal systems, (whereas bacterial hosts are not), it issignificant that marker systems and regulating systems can be providedwithin this context.

Accordingly, one aspect of the invention is a method for obtaininguseful heterologous proteins from vertebrate cell host cultures throughthe use of a polycistronic expression vector which contains sequencescoding for a secondary protein and a desired protein, wherein both thedesired and secondary sequences are governed by the same promoter. Thecoding sequences are separated by translational stop and start signalcodons. The expression of the secondary sequence effects control overthe expression of the sequence for the desired protein, and thesecondary protein functions as a marker for selection of transfectedcells. The invention includes use of secondary sequences having eitheror both of these effects.

In other aspects, the invention concerns the genetic expression vectorssuitable for transfecting vertebrate cells in order to produce thedesired heterologous peptide, the cell culture produced by thistransfection, and the polypeptide produced by this cell culture.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the construction of an expression vector for HBsAg,pE342.HS94.HBV.

FIG. 2 shows the construction of an expression vector for DHFR,pE342.D22.

FIG. 3 shows the construction of an expression vectors for DHFR andHBsAg, pE342.HVB.D22 and pE342.HBV.E400.D22.

DETAILED DESCRIPTION AND DESCRIPTION OF THE PREFERRED EMBODIMENT

A. Definitions

As used herein,

"Plasmids" includes both naturally occurring plasmids in bacteria, andartificially constructed circular DNA fragments.

"Expression vector" means a plasmid which contains at least the fouressential elements set forth hereinabove for the expression of theheterologous peptide in a host cell culture.

"Heterologous protein" means a protein or peptide which is not normallyproduced by, or required for the viability of, the host organism.

"Desired protein" means a heterologous protein or peptide which themethod of the invention is designed to produce.

"Secondary peptide" means the protein or peptide which is not theheterologous peptide desired as the primary product of the expression inthe host cell, but rather a different heterologous peptide which, byvirtue of its own characteristics, or by virtue of the characteristicsof the sequence coding for it is capable of "marking" transfection bythe expression vector and/or regulating the expression of the primarilydesired heterologous peptide.

The peptide sequence may be either long or short ranging from about 5amino acids to about 1000 amino acids. The conventional distinctionbetween the words peptide and protein is not routinely observed in thedescription of the invention. If the distinction is to be made, it willbe so specified.

"Primary sequence" is the nucleotide sequence coding for the desiredpeptide, and

"Secondary Sequence" means a sequence of nucleotides which codes for thesecondary peptide.

"Transfection" of a host cell means that an expression vector has beentaken up by the host cell in a detectable manner whether or not anycoding sequences are in fact expressed. In the context of the presentinvention, successful transfection will be recognized when anyindication of the operation of this vector within the host cell isrealized. It is recognized that there are various levels of successwithin its context. First, the vector's coding sequence may or may notbe expressed. If the vector is properly constructed with inclusion ofpromoter and terminator, however, it is highly probable that expressionwill occur. Second, if the plasmid representing the vector is taken upby the cell and expressed, but fails to be incorporated within thenormal chromosomal material of the cell, the ability to express thisplasmid will be lost after a few generations. On the other hand, if thevector is taken up within the chromosome, the expression remains stablethrough repeated replications of the host cell. There may also be anintermediate result. The precise details of the manner in whichtransfection can thus occur are not understood, but it is clear that acontinuum of outcomes is found experimentally in terms of the stabilityof the expression over several generations of the host culture.

B. A Preferred Embodiment of the Desired Peptide

In a preferred specific embodiment, exemplary of the invention herein,the primary genetic sequence encodes the hepatitis B-surface antigen(HBsAg). This protein is derived from hepatitis B virus, the infectiveagent of hepatitis B in human beings. This disease is characterized bydebilitation, liver damage, primary carcinoma, and often death. Thedisease is reasonably widespread especially in many African and Asiancountries, where many people are chronic carriers with the potential oftransmitting the disease pandemically. The virus (HBV) consists of a DNAmolecule surrounded by a nuclear capsid, in turn surrounded by anenvelope. Proteins which are associated with the virus include thesurface antigen (HBsAg), a core antigen, and a DNA polymerase. The HBsAgis known to produce antibodies in infected people. HBsAg found in theserum of infected individuals consists of protein particles whichaverage ca. 22 nanometers in diameter, and are thus called "22 nanometerparticles". Accordingly, it is believed that the HBsAg particle would bean effective basis for a vaccine.

C. A Preferred Embodiment of the Secondary Peptide

It has been recognized that environmental conditions are often effectivein controlling the quantity of particular enzymes that are produced bycells under certain growth conditions. In the preferred embodiment ofthe present invention, advantage is taken of the sensitivity of certaincells to methotrexate (MTX) which is an inhibitor of dihydrofolatereductase (DHFR). DHFR is an enzyme which is required, indirectly, insynthesis reactions involving the transfer of one carbon units. Lack ofDHFR activity results in inability of cells to grow except in thepresence of those compounds which otherwise require transfer of onecarbon units for their synthesis. Cells lacking DHFR, however, will growin the presence of a combination of glycine, thymidine and hypoxanthine.

Cells which normally produce DHFR are known to be inhibited bymethotrexate. Most of the time, addition of appropriate amounts ofmethotrexate to normal cells will result in the death of the cells.However, certain cells appear to survive the methotrexate treatment bymaking increased amounts of DHFR, thus exceeding the capacity of themethotrexate to inhibit this enzyme (2, 3, 4). It has been shownpreviously that in such cells, there is an increased amount of messengerRNA coding for the DHFR sequence. This is explained by assuming anincrease in the amount of DNA in the genetic material coding for thismessenger RNA. In effect, apparently the addition of methotrexate causesgene amplification of the DHFR gene. Genetic sequences which arephysically connected with the DHFR sequence although not regulated bythe same promoter are also amplified (1, 8, 9, 10). Consequently, it ispossible to use the amplification of the DHFR gene resulting frommethotrexate treatment to amplify concomitantly the gene for anotherprotein, in this case, the desired peptide.

Moreover, if the host cells into which the secondary sequence for DHFRis introduced are themselves DHFR deficient, DHFR also serves as aconvenient marker for selection of cells successfully transfected. Ifthe DHFR sequence is effectively connected to the sequence for thedesired peptide, this ability serves as a marker for successfultransfection with the desired sequence as well.

D. Vector Construction Techniques Employed (Materials and Methods)

The vectors constructed in the Examples set forth in E are constructedby cleavage and ligation of isolated plasmids or DNA fragments.

Cleavage is performed by treating with restriction enzyme (or enzymes)in suitable buffer. In general, about 20 μg plasmid or DNA fragmentsrequire about 1-5 units of enzyme in 200 μl of buffer solution.(Appropriate buffers for particular restriction enzymes are specified bythe manufacturer.) Incubation times of about 1 hour at 37° C. areworkable. After incubations, protein is removed by extraction withphenol and chloroform, and the nucleic acid recovered from the aqueousfraction by precipitation with ethanol.

If blunt ends are required, the preparation is treated for 15 minutes at15° with 10 units of Polymerase I (Klenow), phenol-chlorform extracted,and ethanol precipitated.

Size separation of the cleaved fragments is performed using 6 percentpolyacrylamide gel described by Goeddel, D., et al., Nucleic Acids Res,8: 4057 (1980) incorporated herein by reference.

For ligating approximately equimolar amounts of the desired components,suitably end tailored to provide correct matching are treated with about10 units T4 DNA ligase per 0.5 μg DNA.

E. Detailed Description of a Preferred Embodiment

In general, the expression vector suitable for the present invention isconstructed by adaptation of gene splicing techniques. The startingmaterial is a naturally occurring bacterial plasmid, previouslymodified, if desired. A preferred embodiment of the present inventionutilizes a pML plasmid which is a modified pBR 322 plasmid preparedaccording to Lusky, M. et al. Nature 239: 79 (1981) which is providedwith a single promoter, derived from the simian virus SV-40 and thecoding sequence for DHFR and for HBsAg.

In the construction, the promoter (as well as a ribosome bindingsequence) is placed upstream from the coding sequence coding for adesired protein and one coding for a secondary protein. A singletranscription termination sequence is downstream from both. At the endof the upstream code sequence is placed a translational stop signal; atranslational start signal begins the downstream sequence. Thus,expression of the two coding sequences results in a single mRNA strand,but two separate mature proteins.

In a particularly preferred embodiment, the sequence coding for thesecondary peptide is downstream from that coding for the desiredpeptide. Under these circumstances, procedures designed to select forthe cells transformed by the secondary peptide will also select forparticularly enhanced production of the desired peptide.

F. Examples

The following examples are intended to illustrate, but not limit theinvention.

EXAMPLE 1 Vector Containing the HBsAg Sequence, pE342.HS94.HBV

FIG. 1 shows the construction of the HBsAg plasmid.

The 1986 bp Eco R1-BgIII fragment which spans the surface antigen genewas isolated from the HBV viral genome cloned with pBR322 as describedby Liu, et al., DNA, 1: 213 (1982), incorporated herein by reference.This sequence was ligated between the Eco RI and Bam HI sites of pML, apBR322 derivative which lacks sequences inhibitory to its replication insimian cells, as described by Lusky, et al., Nature, 293: 79 (1981),incorporated herein by reference. Into the single Eco RI site of theresulting plasmid was inserted the 342 bp origin fragment of SV40obtained by Hind III Pvu II digestion of the virus genome, which hadbeen modified to be bounded by Eco RI restriction sites resulting inp342E (also referred to as pHBs348-E) as described by Levinson et al.,patent application Ser. No. 326,980, filed Dec. 3, 1981, which is herebyincorporated by reference. The 5' nontranslated leader region of HBsAgwas removed by treatment with Eco R1 and with Xba, and the analogous 150bp Eco RI-Xba fragment of a hepatitis expression plasmid pHS94 Liu, etal, (supra) was inserted in its place to create pE342.HS94.HBV.

(As described by Liu, et al, pHS94 contains the translational startcodon of the authentic HBsAg gene, but lacks all 5' nontranslatedmessage sequences. The levels of expression of both the authentic EcoRI-Bgl II and pHS94 derived equivalent under control of the SV40 earlypromoter as described above are equivalent and are interchangeablewithout affecting the performance of the plasmid.)

EXAMPLE 2 Vector Containing the DHFR Sequence, pE342.D22

A plasmid carrying DHFR as the only expressable sequence is pE348.D22,the construction of which shown in FIG. 2.

The 1600 bp Pst I insert of the DHFR cDNA plasmid DHFR-11 Nunberg, etal., Cell, 19: 355 (1980) was treated with the exonuclease Bal 31 inorder to remove the poly G:C region adjacent to the Pst I sites,digested with Bgl II and the resulting fragments of approximately 660 bpisolated from gels. The Bal31-Bgl II digested cDNA was ligated into apBR322 plasmid derivative containing a Bgl II site. (Following digestionof pBR322 with Hind III, the plasmid fragment was filled in using KlenowDNA polymerase in the presence of the four deoxynucleotidetriphosphates, and subcut with Bgl II.) The resulting plasmid,pDHFR-D22, has an EcoR I site situated 29 bp upstream of the fusion sitebetween pBR322 and the 5' end of the DHFR cDNA. The EcoR I-Bgl IIfragment encompassing the coding sequences of the cDNA insert was thenexcised from pDHFR-D22 and ligated to EcoR I-BamH I digested pE342.HBV(Example 1), creating the DHFR expression plasmid pE342.D22.

EXAMPLE 3 Vectors Containing Both DHFR and HBsAg Sequences

Two such vectors were constructed, pE342.HBV.D22 containing apolycistron wherein the DHFR gene is downstream from the HBsAg gene, andpE342.HBV.E400.D22, (FIG. 3) in which the genes coding for DHFR andHBsAg are not polycistronic.

A. pE342.HBV.D22 was constructed by ligating the EcoR I-Taq I fragmentof cloned HBV DNA (Liu, et al. (supra)), to EcoR I-Cla I digestedpE342.D22.

B. This plasmid was further modified by fusing an additional SV40 earlypromoter between the Bgl II site and the Cla I site of the DHFR insertof pE342.HBV.D22, creating pE342.HBV.E400.D22.

HBV viral DNA contains a single Taq I site 20 bp beyond the Bgl II sitethat was used to generate the EcoR I-Bgl II fragment encompassing thesurface antigen gene. Thus, EcoR I and Taq I digestion of cloned HBVviral DNA results in a fragment of ˜2000 bp spanning the surface antigengene, and containing a single Bgl II site (1985 bp from the EcoR I site,(Liu, et al (supra)). (The ends of DNA fragments Taq I and Cla Igenerated by digestion are cohesive, and will ligate together).

The Cla I site is regenerated; thus pE342.HBV.D22 contains both a Bgl IIand Cla I site, which are situated immediately in front of the DHFRcoding sequences.

An SV40 origin bounded by restriction sites cohesive with the Bgl II andCla I sites of pE342.HBV.D22 was constructed by digesting SV40 DNA withHpa II, filling in as described above, and subcutting with Hind III. A440 bp fragment spanning the origin was isolated. This was ligated, in atripartite ligation, to the 4000 bp pBR322 fragment generated by HindIII and BamH I digestion, and the 1986 bp fragment spanning the surfaceantigen gene generated by digesting the cloned HBV viral DNA with EcoRI, filling in with Klenow DNA polymerase 1, subdigesting with Bgl II,and isolating on an acrylamide gel. Ligation of all three fragments isachievable only by joining of the filled in Hpa II with EcoR I, the twoHind III sites with each other and the Bgl II with BamH I. Thus when theresulting plasmid is restricted with ClaI and BamH I, a 470 bp fragmentis obtained which contains the SV40 origin. This fragment is insertedinto the Cla I and Bgl II sites of pE342. HBV.D22, (paragraph A)creating pE342.HBV.E400.D22 (FIG. 3).

EXAMPLE 4 Transfection of Host Cells

The host cells herein are vertebrate cells grown in tissue culture.These cells, as is known in the art, can be maintained as permanent celllines prepared by successive serial transfers from isolated normalcells. These cell lines are maintained either on a solid support inliquid medium, or by growth in suspensions containing support nutrients.

In the preferred embodiment, CHO cells, which were deficient in DHFRactivity are used. These cells are prepared and propagated as describedby Urlaub and Chasin, Proc. Natl. Acad. Sci. (USA), 77: 4216 (1980),which is incorporated herein by reference.

The cells are transfected with 5 mg of desired vector as prepared aboveusing the method of Graham and Van Der Eb, Virology, 52: 456 (1978)incorporated herein by reference.

The method insures the interaction of a collection of plasmids with aparticular host cell, thereby increasing the probability that if oneplasmid is absorbed by a cell, additional plasmids would be absorbed aswell. Accordingly, it is practicable to introduce both the primary andsecondary coding sequences using separate vectors for each, as well asby using a single vector containing both sequences.

EXAMPLE 5 Growth of Transfected Cells and Expression of Peptides

The CHO cells which were subjected to transfection as set forth abovewere first grown for two days in non-selective medium, then the cellswere transferred into medium lacking glycine, hypoxanthine, andthymidine, thus selecting for cells which are able to express theplasmid DHFR. After about 1-2 weeks, individual colonies were isolatedwith cloning rings.

Cells were plated in 60 or 100 mm tissue culture dishes at approximately0.5×10⁶ cells/dish. After 2 days growth, growth medium was changed.HBsAg was assayed 24 hours later by RIA (Ausria II, Abbott). Cells werecounted and HBsAg production standardized on a per cell basis. 10-20random colonies were analyzed in this fashion for each vector employed.

In one example of the practice of the invention, the following resultswere obtained:

    __________________________________________________________________________                 Transfectional                                                                efficiency of                                                                            HBsAg production; ng/10.sup.6 cells/day                            Dhfr.sup.- (Percent of colonies in given range)                  Vector       (colonies/ug/10.sup.6 cells)                                                             0   0-10                                                                             10-100                                                                            100-500                                                                           500-1500                                                                           >1500                             __________________________________________________________________________    pE342.D22    935        100 0  0   0   0    0                                 pE342.HS94   <.2        1   1  1   1   1    1                                 pE342.D22 + pE342.HS94                                                                     340        0   50 30  20  0    0                                 pE342.HBV.D22                                                                               20        0   0  0   0   55   45                                pE342.HBV.E400.D22                                                                         510        0   17 17  58  8    0                                 __________________________________________________________________________

The production of surface antigen in several of the highest expressingcell lines has been monitored for greater than 20 passages and isstable. The cells expressing the surface antigen remain attached to thesubstratum indefinitely and will continue to secrete the large amountsof surface antigen as long as the medium is replenished.

It is clear that the polycistronic gene construction results inisolation of the cells producing the highest levels of HBsAg. 100percent of colonies transformed with pE342.HBV.D22 produced over 500ng/10⁶ cells/day whereas 92 percent of those transformed with thenon-polycistronic plasmid pE342.HBV.E400.D22 produced less than thatamount. Only cells from the polycistronic transfection demonstratedproduction levels of more than 1500 ng/10⁶ cells/day.

EXAMPLE 6 Treatment with Methotrexate

The surface antigen expressing cell lines are inhibited by methotrexate(MTX), a specific inhibitor of DHFR at concentrations greater than 10nM. Consistent with previous studies on the effects of MTX on tissueculture cells, occasional clones arise which are resistent to higherconcentrations (50 nM) of MTX at a frequency of approximately 10⁻⁵.However, these clones no longer produce surface antigen despite theamplification of HBV sequences in the MTX resistant clones. Thus, theHBV gene is amplified, though expression falls off in this case. Thissuggests that further production of surface antigen may be lethal to thecell.

EXAMPLE 7 Recovery of Desired Peptide

The surface antigen produced is in the form of a particle, analogous tothe 22 nm particle observed in the serum of patients infected with thevirus. This form of antigen has been shown to be highly immunogenic.When the cells are grown in medium lacking calf serum or othersupplements, approximately 10 percent of the protein contained in themedium is surface antigen and this protein can be isolated by methodsknown in the art. The surface antigen comigrates on SDS-polyacrylamidegels with the 22 nm particle derived protein.

REFERENCES

1. Wigler, M., et al, Proc. Natl. Acad. Sci., 77: 3567 (1980)

2. Schimke, Robert T., et al, Science, 202: 1051 (1978)

3. Biedler, J. L., et al, Cancer Res., 32: 153 (1972)

4. Chang, S. E., et al, Cell, 7: 391 (1976)

5. Fischer, G. A., Biochem Pharmacol., 11: 1233 (1962)

6. Sirotnak, F. M., et al, Cancer Res., 28: 75 (1968)

7. Flintoff, W. F., et al, Somat. Cell. Genet., 2: 245 (1976)

8. Christman, J., et al., Proc. Natl. Acad. Sci., 79: 1815 (1982)

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We claim:
 1. An expression vector capable of expressing in a vertebratehost cell culture a desired protein and a secondary protein, whichvector comprises a DNA sequence encoding for a desired protein and a DNAsequence encoding for a secondary protein wherein both said DNAsequences are operably linked to the same promoter sequence andseparated by translational stop and start codons.
 2. The expressionvector of claim 1 wherein the coding sequence for the secondary proteinencodes for DHFR.
 3. The expression vector of claim 1 wherein thepromoter sequence is the early promoter derived from SV40.
 4. Vertebratecells transformed with the vector of claim
 1. 5. The expression vectorof claim 1 wherein the coding sequence encoding desired protein encodesHBsAg.
 6. A vertebrate host cell culture expression vector capable ofexpressing in a vertebrate host cell culture a desired protein and asecondary protein which vector contains the coding sequences for adesired heterologous protein and for a secondary protein, both operablylinked to the same promoter, and separated by translational stop andstart codons, wherein the sequence coding for the secondary protein isdownstream from the sequence coding for the desired protein.
 7. Theexpression vector of claim 6 wherein the coding sequence for thesecondary protein encodes for DHFR.
 8. The expression vector of claim 6wherein the coding sequence encoding desired protein encodes HBsAg.